Regulation of mitochondrial protein synthesis, and the targeting of mitochondrial gene products to their correct destinations in the inner membrane, are essential for normal metabolism in virtually all eukaryotes. The proposed project will study these processes in Saccharomyces cerevisiae, taking advantage of expertise in manipulating the yeast mitochondrial genome and the successful development of synthetic mitochondrial selectable markers and reporter genes. Studies using these reporter genes have demonstrated that membrane-bound mRNA- specific translational activators play key roles in localizing translation of the yeast mitochondrial COX2 and COX3 mRNAs to the inner membrane, and in their regulation. They have also opened the door to in vivo analysis of the translocation process that exports hydrophilic domains of Cox2p to the intermembrane space. One aim of this project will be to characterize the as-yet-unidentified export translocation apparatus, relying primarily on a novel selective scheme based on the export of a mitochondrially coded arginine biosynthetic enzyme fused to the C-terminus of Cox2p. The second aim will be to study the role, suggested by preliminary work, of the pre- Cox2p leader peptide and/or the mRNA sequence encoding it in posttranscriptional regulation of COX2 expression. This phenomenon could provide a mechanism for cytochrome oxidase assembly to feedback- regulate subunit synthesis. The existence of membrane-bound mRNA- specific translational activators could facilitate such controls and/or allow advantageous topological distinctions between the sites where different subunits are synthesized. To explore these possibilities the third aim will be to construct stable strains in which the subunit coding sequences are placed in noncognate loci in mtDNA, thus rearranging the wild-type coupling of mRNA 5'-UTLs, containing translational activator targets, with structural genes. The final aim will be to continue to characterize the mechanism of mitochondrial translation initiation, a process that cannot be studied in vitro. Genes encoding proteins, and/or RNAs, that interact with functional sites in the COX2 mRNA 5'-UTL will be identified, and the structure of translational activation complexes examined. Activators from divergent yeast species will be characterized to reveal conserved functional domains and generate tools with which to ask whether such activators are present in other groups of eukaryotes. To identify new translational regulatory functions, genetic alterations that increase expression of mitochondrial reporter genes will be sought.